The long-term objective of this project is to understand the life cycle of dsDNA viruses through study of the molecular mechanisms by which their virions assemble and function. The group of viruses chosen for this study, the tailed bacteriophages, have direct human medical importance since they are the most abundant 'life form' on Earth and they have huge importance in bacterial pathogenicity and microbial ecology. They also serve as very important models for similar processes in less experimentally accessible eukaryotic viruses such as Herpesviruses, Iridopoxviruses and Adenoviruses. Thus the proposed research is relevant to human health. The P22 bacteriophage and its close relatives that are utilized in this project constitute one of the most genetically and biochemicall well-characterized virus systems, and so are ideal for obtaining new knowledge about the assembly and function of virus particles. This type of virus first assembles a protein shell, calle a procapsid, and then inserts the dsDNA chromosome into this shell to form a virion. The P22 procapsid contains three critical polypeptides, a coat protein that forms the external shell, an internal scaffolding protein that is required to build the procapsid but leaves before DNA enters, and a portal protein that forms a channel through which DNA enters the coat protein shell. A virus-encoded ATP cleavage driven DNA translocase (called 'terminase') interacts with procapsids and is central to the DNA recognition and entry processes. The DNA translocase and the procapsid with its scaffold are central features of the assembly strategy of most if not al large dsDNA viruses; however, many features of their assembly and function remain unknown. The role of the scaffolding protein in procapsid assembly is one focus of this proposal. It guides coat protein's assembly into a closed shell by co-assembling with it, and it recruits other protein into the structure. After co-assembly with coat protein to form procapsids, scaffolding protein is released from the procapsid before the DNA insertion step, and so it functions catalytically in virion assembly. The other focuses of the proposal are aimed at understanding the structure and function of the components of the DNA packaging and injection molecular machines which include the terminase DNA packaging motor and proteins that are injected into cells with the DNA. The aims of the project will be pursued through a combination of genetic, biochemical, biophysical and structural analysis strategies.